Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1998-03-04
2001-10-02
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S528000, C436S529000, C436S531000, C436S533000, C436S535000, C436S538000, C436S541000, C436S542000, C436S807000, C436S808000, C436S809000, C436S810000, C436S501000, C436S824000, C436S518000, C435S006120, C435S007100, C435S007200, C435S007500, C435S007800, C435S004000, C435S173100, C435S007920, C435S007900, C435S973000, C435S975000
Reexamination Certificate
active
06297062
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns a method for separating and detecting biological molecules, utilizing magnetic particles. The present invention fur tier concerns products, and kits used in said method, as well as methods for the preparations of said products.
BACKGROUND OF THE INVENTION
Magnetic particles are used for a variety of separation, purification, and isolation techniques in connection with biological biomolecules. In those techniques, a magnetic particle is coupled to a molecule capable of forming a specific binding (hereinafter “affinity binding”) with a molecule in a biological sample, which is to be isolated, purified or separated. The biological sample is then brought into contact with the magnetic particle and those biological molecules which bind to the magnetic particle are then isolated by application of a magnetic field.
Such magnetic separation techniques have been employed to sort cells, to recover antibodies or enzymes from a solution, to purify proteins using affinity techniques, and to remove unwanted particles from suspension, for example, to remove cancer cells ex vivo, from a cell preparation which is then injected into a patient (Pourfarzaneh, M. et aL, “
The use of magnetizable particles in solid phase immunoassay in methods of biochemical analysis”
28:267-295 (1982)).
U.S. Pat. No. 4,554,088 discloses a process for the preparation of magnetic particles, capable of coupling to a wide variety of molecules. The magnetic particles are then dispersed in an aqueous media without rapid settling, and can conveniently be reclaimed from the media using a magnetic field.
U.S. Pat. No. 4,169,804 discloses a magnetically responsive composite microparticle comprising a magnetically responsive material and a porous solid water-insoluble matrix such as proteinaceous material, polysaccharides and the like. The magnetically responsive material is dispersed throughout the permeable, solid, water insoluble matrix. The microparticles of this U.S. patent, are intended to substitute microparticles in labeling or for the purpose of carrying medicaments and drugs, since due to their magnetic responsiveness, they can be easily retrieved or relocated without the need to employ expensive equipment The microparticles are also used as vectors in chemical reactions, and in determining levels of biological compounds in a sample.
Separation, isolation and purification techniques utilizing magnetic particles, are used to separate or isolate a single species of molecules from a sample at a time, depending on the nature of the molecule coupled to the magnetic bead. Prior art methods, do not disclose methods for separating several molecular species from a sample simultaneously.
U.S. Pat. No. 5,395,498 discloses a method for obtaining molecules which are separated on an electrophoretogram. Said electrophoretogram is then brought into contact with a matrix of magnetic particles having binding specificity to one or more biological molecules present on the electrophoretogram, whereby these molecules bind to the magnetic particles in the matrix. The particles can then be collected on the basis of their magnetic properties, and by this the electrophoretically separated molecules can be obtained. Although this U.S. patent discloses a method suitable for obtaining several different species of biological molecules simultaneously utilizing magnetic means, it concerns only molecules separated on electrophoretograms, and does not concern at all molecules present in a liquid sample.
It would have been highly desirable to provide a method, and means for a simultaneous separation and isolation of several different species of biological molecules from a liquid sample without the step of electrophoretic separation.
Blotting techniques, were initially constructed for transferring DNA patterns from electrophoretic agarose gel to nitrocellulose membrane filters. A modification of the blotting technique termed “dot hybridization method” F. C. Kafatos,
Nuc. Acids. Res.,
7:1541-1552 (1979
)) was developed, in which, instead of being blotted from a gel to the filter, the probe is applied directly to the nitrocellulose filter at a discrete spot. Such a construction allows considerable simplification of the detection procedure and simultaneous screening of a large number of samples. An analogous protocol for antigen-antibody reaction termed “dot immuno binding” was described by Richard Hawkes (
Anal Bio
119:142-147 (1982
)) for applications such as screening of hybridoma clones, where a large number of samples must be screened for a specific antibody, and for a diagnostic procedure by which a large number of antibodies may be assayed simultaneously. A modification of the dot immuno binding termed “dot-ELISA” (dot-enzyme-linked immunosorbent assay) was described by M. G. Pappas (
J. Immun. Mth,
64:205 (1983
)). Dot-ELISA combines the advantages of both the above methods and can be compared in sensitivity to radioimmunoassay.
Commercial equipment for dot immuno-binding such as the Bio-Dot™ and the Bio-Dot SF™ (Slot Format) microfiltration units from Bio-Rad (ISA) provide a reproducible method for binding proteins or nucleic acids present in liquid solutions onto nitrocellulose or Zeta-Probe™ (Bio-Rad, U.S.A.) membranes. The disadvantage of such equipment, and similar equipments, resides in the necessity for manual handling, which requires, for example, manual pipettes to apply the samples onto the membranes.
Biomek 1000™ (Beckman, USA) is a robotic work station which enables automization of procedures for DNA hybridization. It consists of two different devices for creating dots on a filter sheet: a dot-blotting apparatus and a high density replicating (HDR) system designed to allow the replication of a large number of bacteria, phage or DNA samples from a microtiter plate to a single filter. The precision of the Biomek allows to produce samples in a grid format, with high reproducibility. The high density replicating system uses a 96-pin tool in order to transfer the samples from the microtiter plate to the suitable substrate which may be agar or membrane. The equipment is automatically cleaned and sterilized between each successive transfer of 96 samples from the microtiter plate. Samples from up to 36 microtiter plates can be transferred to a single substrate.
The robotic station Biomek 1000™ is adapted for high density spotting (Dramanac et al,
Electrophoresis,
13:566-573 (1992
)).
“Sequencing by hybridization”, refers to a technique for sequencing an unknown stretch of oligonucleotides, by determining whether it can form a mismatch-free hybridization with a series of shorter known sequences, termed “probes”. Technically, sequencing by hybridization (SBH) can be performed in two formats, with either the DNA or the short oligonucleotide probes immobilized to a solid support. In the first format, the probes are labeled by individual detectable labels, and hybridize to a genomic DNA immobilized to the support. In the second format, genomic fragments of appropriate length are appropriately labeled, and for each of them a separate hybridization reaction with immobilized probes is carried out. The first format, is technically carried out by a dot-blot hybridization system using Beckman's commercial HDR system or similar commercially available systems. The second format is carried out by a technically different variant system, capable of spotting synthesized oligonucleotides on glass covered by activated acrylamides, and direct parallel synthesis of oligonucleotide by physical masking, by photolithography, or by a combination of synthesis of oligonucleotides on microbeads and formation of a fixed bead monolayer. A solid support with samples which may be either the short probe oligonucleotides or genomic DNA samples, spaced on a micrometer scale, has been termed “a sequencing chip”.
One of the most severe problems in both formats of sequencing by hybridization technique is the time consuming step of washing of the filters. In order to permit multiple usage of the filters it is nec
Bio-Magnetics Ltd.
Do Pensee T.
Le Long V.
Oliff & Berridg,e PLC
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